1. Field of the Invention
The present invention relates generally to methods for identifying organ-specific proteins that are secreted into the blood. The invention further relates to methods of diagnosis and methods of use of such proteins.
2. Description of the Related Art
The ability to detect the onset of disease very early has been a longtime goal of the diagnostic field. Early detection will in most cases permit the disease to be effectively dealt with. For example, with most cancers, early detection would permit a patient to be cured by conventional therapies (chemotherapy, radiation, surgery). Hence early diagnosis is the cornerstone of dealing effectively with many diseases.
Differentially expressed proteins, particularly proteins found in blood, may serve as biological markers that can be measured for diagnostic (or therapeutic) purposes. Different approaches for measuring blood proteins have been used with varying degrees of success. In particular, two-dimensional (2-DE) gel electrophoresis is widely used for analysis of proteomic patterns in blood and other tissues. However, several limitations restrict its utility in diagnostic proteomics. First, because (2-DE) gels are limited to spatial resolution, it is difficult to resolve large numbers of proteins such as are expressed in the average cell (1,000 to 10,000 proteins) or even worse—blood. High abundance proteins can distort carrier ampholyte gradients in capillary isoelectric focusing electrophoresis (CGE) and result in crowding in the gel matrix of size sieving electrophoretic methods (e.g., the second dimension of (2-DE) gel electrophoresis and CGE), thus causing irreproducibility in the spatial pattern of resolved proteins (see e.g., Corthals, G. L., et al. Electrophoresis, 18:317 (1997). Lopez, M. F., and W. F. Patton, Electrophoresis, 18:338 (1997)). Note, for example, that albumen constitutes about 51% of the blood protein. Indeed, 22 proteins constitute about 99% of the blood protein and most of these will not be useful diagnostic markers—those will be present in the 1% of the remaining proteins that are often hidden by the abundant proteins. High abundance proteins can also precipitate in a gel and cause streaking of fractionated proteins (Corthals, G. L., et al., supra). Variations in the crosslinking density and electric field strength in cast gels can further distort the spatial pattern of resolved proteins. Another problem is the inability to resolve low abundance proteins neighboring high abundance proteins in a gel because of the high staining background and limited dynamic range of gel staining and imaging techniques. Limitations with staining also make it difficult to obtain reproducible and quantifiable protein concentration values, with average standard variations in relative protein abundance between replicate (2-DE) gels reported to be 20% and as high as 45% (Anderson, L. and J. Seilhamer, Electrophoresis, 18:533 (1997)). For example, investigators were only able to match 62% of the spots formed on 3-7 gels run under similar conditions (Lopez, M. F., and W. F. Patton, supra; see also Blomber, A., et al., Electrophoresis, 16:1935 (1995) and Corbett, J. M., et al., Electrophoresis, 15:1205 (1994)). Additionally, many proteins are not soluble in buffers compatible with acrylamide gels, or fail to enter the gel efficiently because of their high molecular weight (see e.g., Ramsby, M., et al., Electrophoresis, 15:265 (1994)).
Thus, a major stumbling block in the diagnostic proteomic analysis of the blood is the high degree of complexity of the blood proteome. Another major challenge is the large dynamic range across which proteins are expressed—about 10e10. This means that one protein may be present at one copy in a given volume, whereas another may be present at 10e10 copies. Additionally, pattern analysis using techniques such as 2-DGE and other similar techniques has been problematic primarily as a result of the irreproducibility of the gel patterns, inability to detect very low abundance proteins, difficulty in quantitating the individual spots (e.g., proteins) that make up a complex proteomic pattern and the inability to identify the individual proteins that constitute the complex pattern. Further, the ability to extend these techniques to easy, consistent, and high throughput diagnostic assays has been extremely limited. Thus, there is a need in the art to provide such diagnostic assays. The present invention provides for methods and assays that fulfill these and other needs.